Method of diagnosing and preventing pneumococcal diseases using pneumococcal neuraminidases

ABSTRACT

A method of providing protection against pneumococcal infection in a subject is disclosed. The method includes steps of administering to the subject a composition that includes combination of three recombinant pneumococcal neuraminidases: NanA, NanB, and NanC of  S. pneumoniae  strains CGSP14, wherein administration of the recombinant pneumococcal neuraminidases elicits an immune response to  S. pneumoniae , and treats the subject. In one embodiment, the method further includes a step of adding adjuvants to enhance the immune response. The method also includes a step of using passive antibodies, wherein said passive antibodies are anti-neuraminidase antibodies generated from neuraminidases-immunized humanized animals: NanA, NanB, and NanC. Meanwhile, this invention also provides a method for the molecular diagnosis of pneumococcal infection.

FIELD OF THE INVENTION

The present invention relates to the development of vaccine for preventing pneumococcal diseases, and to the diagnosis of the pneumococci-infected samples not just from urine but also from blood and pleural effusion in pyothorax. More particularly, this invention relates to a universal protein vaccine against the pneumococcal infection.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae is one of Gram-positive encapsulated diplococci. Pneumococcal infection is a leading infectious cause of the high mortality and morbidity worldwide, especially among young children below two years of age and the elderly over sixty years of age. Globally, pneumococcal infection has been estimated to cause about 1.6 million deaths annually, including 1 million children less than five years old. Even though certain vaccines have been applied to prevent the S. pneumoniae infection, the mortality rate caused by this organism is still ranked the highest. The spectrum of the S. pneumoniae-related diseases includes invasive pneumococcal disease (IPD), such as sepsis and meningitis; lower respiratory infections, such as bacterial pneumonia; and upper respiratory infections, such as acute otitis media (AOM) (Tuomanen et al., 1995).

According to the reports of World Health Organization (WHO) in 2005, acute respiratory tract infections were the major cause of death globally, in which the deaths were chiefly attributable to the S. pneumoniae-associated community-acquired pneumonia (CAP). This threatening issue strongly raises the urgency for both diagnosis and prevention. Although the diagnoses of pneumococci have been developed for decades, we still heavily rely on conventional culture methods that are tedious and time-consuming, to proliferate enough bacteria for specific and sensitive detection. Therefore, based on specific DNA amplification and antigen detection, the tests of non-culture samples from sputum, urine, and blood have been continuously developed over time in order to identify pneumococci as the etiological agent of diseases. However, the consequences of those tests were always unsatisfactory in certain applications. For instance, the application of PCR testing for the diagnosis of IPD has ever shown to be insufficiently sensitive when using blood or urinary samples, and poorly specific when using respiratory samples. To overcome the problem of poor specificity when using sputum samples, recently a dual-PCR testing protocol using pneumococcal lytA and ply as targets has been successfully developed and evaluated.

Another disappointing aspect for diagnosis revealed that only one third of pathogens could be recovered from patient's sputum when using conventional culture methods. In addition, the controversial results lack specificity correlated to CAP because nasopharyngeal carriage of pneumococci could also be found in both healthy individuals and inadequate sputum samples. In addition, the etiological pathogens of CAP tested from blood culture and pleural fluid were specific, but the positive rates were lower (<30%) compared to that from sputum sample. For this reason, the development of antigen detection was applied to compensate the drawback of low specificity. Higher sensitivity of the pleural test compared to pleural cultures indicated that antigen detection for pleural samples rather than pleural culture could be a better application for the CAP study of pneumococcal etiology. Also, the detection of BinaxNOW pneumococcal C-polysaccharide in a urine sample with CAP shows unsatisfied result due to its high rate of false positive.

Currently, there are two kinds of vaccines, 23-valent pneumococcal polysaccharide vaccine (PPV23) and 7-valent pneumococcal conjugate vaccine (PCV7), available for general protection against potential IPD-causative pathogen strains. Vaccine PCV7 can target seven serotypes, including 4, 6B, 9V, 14, 18C, 19F, and 23F. Before the introduction of PCV7, the PCV7-targeted 7 serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) were responsible for about 90% of incidence of IPD in young children in the United States and for more than 60% of those in Europe. After PCV7 vaccination, the cases of IPD in children less than 5 years old declined by 56% in 2001 and by 76% in 2004. In contrast, PPV23 vaccination seems to difficultly reach firm conclusions in clinical effectiveness (around 50-70% effective). Although two doses of PCV7 and following one dose of PPV23 were recommended to broaden protection, the effectiveness of vaccines was significant on the protection of those seven PCV7-covered serotypes rather than others. The results suggested that PPV23 seems not necessary as a boost dose for broadening protection.

At least 93 different polysaccharide (PS) capsules of S. pneumoniae have been verified to be specific serotypes, and further classified to be 46 serogroups. Among all pathogenic pneumoncocci worldwide, serotype 14 and serogroup 6 are predominant. In addition, the majority of IPD is generally caused by about 15 serotypes. However, only a few antimicrobial resistant pneumococcal clones could spread fast. The incidence of antimicrobial resistance of pneumococci varies regionally, and is associated with the spectrum of antibiotic use, population density, the indigenous prevalence of resistant strains, ages and time. Although the resistance patterns have been shown to be different around the world, the predominant serotypes commonly identified are 6A/B, 9V, 14, 19A/F, and 23F. Based on epidemiological study, the nasopharyngeal (NP) carriage of predominant pneumococci has been observed in many young children, indicating that NP carriage may play an important role in pneumococcal transmission, especially for antibiotic-resistant strains.

Despite effective reduction of the incidence of IPD caused by vaccine serotypes in both children and adults due to the usage of the current pneumococcal vaccine PCV7, the mortality rate of pneumococcal disease remains high. After the introduction of PCV7 in 2000, nonvaccine serotype 3 was found to be a significant cause for necrotizing pneumonia in children in Utah, whereas a mucoid serotype 3 was usually reported to cause lung abscess in adults. Serotype 19A has been reported the predominant serotype causing IPD all over the world. In Taiwan, complicated pneumococcal pneumonia still remains a clinically intricate problem, and its significant association with the clonal spread of CC320 within serotype 19A was noteworthy recently in Taiwan. Besides pneumococcal serotypes 3 and 19A, other nonvaccine serotypes, including 1, 5, 6A, and 7F, were also common causes for IPD around the world (Grijalva and Pelton, 2011). A second-generation 13-valent pneumococcal conjugate vaccine (PCV13) was therefore developed to address this new global issue of pneumococcal infection in 2010 (Grijalva and Pelton, 2011).

Hemolytic uremic syndrome (HUS), one of the most severe complications of IPD, mainly occurs in children, and it is also associated with hemolytic anemia, thrombocytopenia, and acute renal failure. This disorder, usually occurring in healthy young children, is one of the most common causes of acute renal failure in pediatric patients. Management of the pneumococcal HUS primarily includes an intensive antimicrobial therapy and the dialysis and transfusion of washed RBC, platelets and plasma. Most cases of HUS are reported by an acute gastroenteritis related to Escherichia coli (O157:H7), and often show good prognosis with recovery of renal function. However, the mortality rate of patients with pneumococcal HUS was high in early reports. Of the 14 cases recently reported from USA, 1 (7%) died and 4 (29%) developed chronic kidney disease.

S. pneumoniae encodes many virulence factors, but only the secreted neuraminidase A (NanA) was reported to be attributed to HUS. Neuraminidase cleaves N-acetylneuraminic acid (sialic acid) residues on red blood cells (RBC), platelets and endothelial cells, and the results may lead to the exposure of the Thomsen-Friedenrich antigen (T antigen), and allow the circulating anti-T antigen antibodies to react with the exposed T antigen on cells. The role of neuraminidase(s) in pneumococcal diseases is illustrated based on the fact that pneumococci produce two or three distinct neuraminidases, which are NanA, NanB, and NanC. All three neuraminidases have typically signal peptides for secretion, wherein NanA, unlike NanB and NanC, contains a C-terminal cell surface anchorage domain. NanA and NanB expose host cell surface receptors for pneumococcal adherence by cleaving sialic acid from the glycans and mucin of cell surface, and thereby it promotes the pneumococcal colonization on the upper respiratory tract. In in vivo study, a NanA mutant was cleared from the nasopharynx, trachea, and lungs within 12 hours postinfection, while a NanB mutant persisted but did not increase in either the nasopharynx, trachea, or lungs. However, the role of NanC remains unknown.

The nonvaccine serotypes have been emerging after the use of vaccines. Moreover, nothing worse than the fact that nonvaccine strains usually displayed increase antimicrobial resistance and virulence. This is the reason why the issue of pneumococcal infection remains to be a global public health challenge. Thus, continued efforts to develop new diagnostic methods and to develop vaccines with expanded or universal coverage, such as a universal protein vaccine, are critically required for the better control of the pneumococcal infections.

SUMMARY OF THE INVENTION

Based on our finding that S. pneumoniae isolates causing HUS, were mostly found to produce all of the three neuraminidases, including NanA, NanB, and NanC, we designed three primer sets to detect and clone these pneumococcal neuraminidase genes in this invention. The three recombinant neuraminidases combined together serve as an ideal vaccine candidate because it presents the best protective efficacy against pneumococcal infection in mice, compared to the others. In one embodiment, the present invention provides a method of molecular detection of pneumococcal diseases by PCR in a S. pneumoniae-infected sample to amplify three neuraminidase genes based on the sequences of the three neuraminidase genes of S. pneumoniae strain CGSP14. In another embodiment, the present invention provides a method of generating immunization in humans and animals against S. pneumoniae infection using a composition comprising the three recombinant neuraminidases, including NanA, NanB, and NanC.

In still another embodiment, the present invention provides protection against S. pneumoniae infection using three pneumococcal neuraminidases as antigens in active immunization, and/or using anti-neuraminidase antibodies for passive immunization. In a further embodiment, the present invention provides a method of detecting inhibition of a neuraminidase activity by antibody or antiserum using flow cytometry. In still a further embodiment, the present invention is able to detect the presence of any of the three neuraminidases in the S. pneumoniae-infected samples using the anti-neuraminidase antibodies generated from neuraminidases-immunized humanized animals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Percentage of neuraminidase genes nanA, nanB and nanC among Streptococcus pneumoniae isolates derived from HUS patients and non-HUS controls (nanC 89% vs. 41% *p<0.005).

FIG. 2: Schematic diagram of the recombinant clones. Each PCR-amplified amplicon of neuraminidase genes (including nanA, nanB, and nanC), which sequences were based on the genomic sequence of S. pneumoniae strain CGSP14, was cloned into an expression vector pET29b through restriction enzyme digest by KpnI and XhoI. Seq. ID (from No. 1 to No. 6) indicates individual sequences among those genes and clones.

FIGS. 3A-3D: Thomsen-Friedenrich antigen (TA) exposure on cells. PNA lectin binding was used to detect the TA by flow cytometry. Numbers indicate fluorescence counts of samples, which are untreated cells (black), NanA-treated (white), NanB-treated (grey), and NanC-treated (hatched). NanA (0.01 μg), NanB (1 μg) and NanC (1 μg) can expose TA on RBC (Figure A). NanA, NanB and NanC (all were 1 μg) can expose TA on A549 (Figure B) and HK-2 cells (Figure C). Twenty 4 aliquots of PNA lectin labeled RBC used for flow cytometric analysis were incubated at 37° C. and observed under microscope to verify agglutination. Agglutination of RBC was observed when treated with NanA, NanB and NanC (0.1 μg) (Figure D).

FIG. 4: Confirmation of mouse polyclonal antisera against neuraminidase(s) by enzyme-linked immunosorbent assay (ELISA). Mouse post-immune (grey box) antisera against neuraminidase(s) antigens from different combinations, including individual neuraminidase (NanA, NanB, or NanC), neuraminidase A+B (NanA+NanB), and neuraminidase A+B+C (NanA+NanB+NanC), were tested by ELISA, while the sera from pre-immune (black box) and negative control (only PBS plus Freund's adjuvant without antigen; white box) were also examined. The antigen-antibody interactions were quantified by using the peroxidase-conjugated goat anti-mouse IgG (Sigma) as a secondary antibody and tetramethylbenzidine/peroxide (R&D Systems, Minneapolis, Minn., USA) as a color-developing substrate under the analysis of ELISA reader with the maximum absorbance band at a wavelengthh of 405 nenometer (EMax, Molecular Devices, Sunnyvale, Calif. 94089 USA). The value was presented by the logarithm of the value on y-axis.

FIG. 5: Vaccination tests. The individual or combination of three neuraminidases, NanA, NanB and NanC with 10 μg of each enzyme, were applied as antigens to immunize mouse (BALB/C, one month old) four times at 2-week interval, while PPV23 vaccine and a negative control (PBS+Freund's adjuvant) were taken for comparison. Thereafter, S. pneumoniae serotype 3 (3×10³ cfu) was used to challenge those neuraminidase-immunized mice. Freund's complete adjuvant for the first time immunization and Freund's incomplete adjuvant for the last three immunizations were used with the ratio of 1:1 to the antigen(s). Mice survival rate (%) was determined during 14 days feeding after four times neuraminidase immunization and a subsequent S. pneumoniae challenge. N is the number of mice for test.

FIGS. 6A-6C: Inhibition of neuraminidase activity by the anti-serum raised from neuraminidase-immunized rabbit. Neuraminidase-mediated TA antigen exposure presented on RBC cells was quantified by flow cytometry analysis, where FITC-labeled PNA lactin was used for the recognition of TA antigen. Prior to the quantification of TA exposure, an individual neuraminidase (including NanA, NanB, and NanC) was individually added for the treatment with different anti-neuraminidase anti-sera (30 μg/mL), including purified anti-NanC antiserum. (Figure A) NanA added for the treatment of rabbit anti-neuraminidase anti-serum were 1 μg (black bars), 0.1 μg (white bars), and 0.01 μg (grey bars). (Figure B) NanB added for the treatment of rabbit anti-NanB anti-serum was 1 μg (white bars). (Figure C) NanC added for the treatment of rabbit anti-NanC anti-serum was 1 μg (grey bars). * indicates p<0.05, when compared to the controls (only neuraminidase addition without serum treatment; neuraminidase addition with pre-immune serum treatment).

FIGS. 7A-7C: Inhibition of neuraminidase activity by the anti-serum raised from neuraminidase-immunized mouse. Neuraminidase-mediated TA antigen exposure presented on RBC cells was quantified by flow cytometry analysis, where FITC-labeled PNA lactin was used for the recognition of TA antigen. Prior to the quantification of TA exposure, an individual neuraminidase (including NanA, NanB, and NanC) was added for the treatment with specific anti-neuraminidase anti-sera (30 μg/mL). (Figure A) NanA added for the treatment of mouse anti-NanA anti-serum was 0.1 μg. (Figure B) NanB added for the treatment of mouse anti-NanB anti-serum was 1 μg. (Figure C) NanC used for the treatment of mouse anti-NanC anti-serum was 1 μg. * indicates p<0.05, when compared to the controls (only neuraminidase addition without serum treatment; neuraminidase addition with pre-immune serum treatment).

DETAILED DESCRIPTION OF THE INVENTION

The detailed description set forth below is intended as a description of the presently exemplary device provided in accordance with aspects of the present invention and is not intended to represent the only forms in which the present invention may be prepared or utilized. It is to be understood, rather, that the same or equivalent functions and components may be accomplished by different embodiments that are also intended to be encompassed within the spirit and scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described can be used in the practice or testing of the invention, the exemplary methods, devices and materials are now described.

All publications mentioned are incorporated by reference for the purpose of describing and disclosing, for example, the designs and methodologies that are described in the publications which might be used in connection with the presently described invention. The publications listed or discussed above, below and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

A. Detection of Neuraminidase Genes nanA, nanB and nanC of S. Pneumoniae

NanA and NanB have been considered to be virulence factors of S. pneumoniae; however, NanC remains poorly understood. The nanC gene was found in the genome of a serotype 14 strain that was isolated from a child with HUS. In this invention, we confirmed that the S. pneumoniae neuraminidase genes nanC as well as nanA and nanB are important virulence factors. Three primer sets for polymerase chain reaction (PCR) are designed for the detection and cloning of the neuraminidase genes that are nanA (Seq. ID No. 1), nanB (Seq. ID No. 2), and nanC (Seq. ID No. 3), based on the genomic sequence of S. pneumoniae strain CGSP14 with NCBI accession number NC_(—)010582. The detection comprises those pneumococcal isolates, especially for invasive pneumococcal diseases, including HUS.

Primer Design to Amplify nanA, nanB and nanC

Three primer sets used for PCR-amplification of nanA, nanB and nanC are designed with two purposes: one is provided for gene detection, and the other for gene cloning into an expression vector as described later.

-   -   1. For nanA amplification based on Seq. ID No. 1:

NanA-ATG-Kpn1: 5′-AGATCTGGGTACC ATGTCTTATTTCAGAA ATCG NanA-TAA-Xho1: 5′-TGGTG CTCGA G TTGTTCTCTCTTTTTCCCT A

-   -   -   The expected size of amplicon is 2964 bp long.

    -   2. For nanB amplification based on Seq. ID No. 2:

NanB-ATG-Kpn1: 5′-AGATCTGGGTACC ATGAATA AAAGAGGTCTTTA NanB-TAA-Xho1: 5′-TGGTG CTCGA G TTTTGTTAA ATCATTAATT TC

-   -   -   The expected size of amplicon is 2115 bp long.

    -   3. For nanC amplification based on Seq. ID No. 3:

NanC-ATG-Kpn1: 5′-AGATCTGGGTACC ATGAAAAAAAAT  ATTAAACA NanC-TAA-Xho1: 5′-TGGTG CTCGA G ATTCTTTTTCAGATCTTCAA

-   -   The expected size of amplicon is 2244 bp long.

In these primer sets, the bold sequences based on the neuraminidase genes are designed for the cloning of full length of genes; the underlined sequences GGTACC and CTCGAG are the KpnI and XhoI recognition sites, respectively, which are built in for cloning into an expression vector; and the plain sequences are extra-sequences which are generated for efficient digests by KpnI and XhoI.

Detection of Neuraminidase Genes nanA, nanB and nanC in Pneumococcal Isolates

The clinical data related to S. pneumoniae infection from Chang Gung Memorial Hospital (CGMH), Taoyuan, Taiwan were compiled in the study of this invention. The invasive pneumococcal disease (IPD) cases are defined as the isolates of S. pneumoniae from normally sterile sites, such as blood, cerebrospinal fluid, or pleural fluid. Patients hospitalized with HUS, associated with an IPD between January 2006 and December 2009, were children less than 18 years old. HUS is defined according to the definition of Centers for Disease Control and Prevention (CDC, 1997). For HUS diagnosis, coagulation studies were examined, and the presence of normal fibrinogen was used to rule out disseminated intravascular coagulopathy. HUS patients enclosed were confirmed for Thomsen-Friedenrich antigen (TA) activation by the peanut (Arachis hypogaea) lectin agglutination method.

In our study, 18 S. pneumoniae isolates from patients with HUS and 54 from non-HUS patients were collected for detecting the neuraminidase genes by PCR using the primer sets designed as the above section. S. pneumoniae intrinsically carry nanA and nanB, as 100% of isolates from both groups have the two genes; however, relative to 16 (89%) of the HUS isolates that harbor nanC, only 22 (41%) isolates from the 54 controls carry the gene (P=0.002) (FIG. 1).

Among the total of 72 S. pneumoniae isolates examined in this invention, 72% ( 21/29) of the serotype 14 isolates contained nanC and 48% ( 14/29) of the patients infected by this serotype caused necrotizing pneumonia. Furthermore, 56% ( 5/9) of the serotype 3 isolates contained nanC and 44% ( 4/9) caused necrotizing pneumonia. Although 60% ( 6/10) of the serotype 6B and 71% ( 5/7) of the 23F also contained nanC, the two serotypes less commonly had necrotizing pneumonia and HUS. In contrast, 19F and its two allele MLST variant 19A seldom contained nanC (only 1 19F), but 38% (⅜) of the patients infected by 19A and 22% ( 2/9) by 19F had necrotizing pneumonia. The difference showed marginally significant (P=0.051) between HUS isolates and those specifically from necrotizing pneumonia patients.

Given the fact that almost all patients with HUS caused necrotizing pneumonia, the result suggests that NanC should be a virulence factor for necrotizing pneumonia as well as for HUS. We conclude that nanC gene is one of important microbe factors for necrotizing pneumonia and HUS caused by S. pneumoniae serotypes, not just by serotype 14 as mentioned previously.

B. Biofunctional Assays of Neuraminidase NanA, NanB And NanC of S. pneumoniae Strains CGSP14

In order to analyze the biofunction of neuraminidases in this invention, the recombinant NanA, NanB, and NanC of S. pneumoniae strains CGSP14 were cloned by using the primer sets as described in the previous section, and also characterized for their features. These biofunctional assays characterized include the exposure of the Thomsen-Friedenrich antigen (TA) and substrate specificity as the follows.

Cloning, Expression, and Purification of Recombinant NanA, NanB, and NanC

Referred to FIG. 2, genes nanA (Sequence ID No. 1), nanB (Seq. ID No. 2), and nanC (Seq. ID No. 3) based on the genomic sequence of S. pneumoniae strain CGSP14 with NCBI accession number NC_(—)010582 were PCR-amplified and cloned into the expression vector pET29b (NOVAGEN, MERCK, Darmstadt, Germany) using KpnI and XhoI as cloning sites; the resulting clones are pET29b-NanA (Seq. ID No. 4), pET29b-NanB (Seq. ID No. 5), and pET29b-NanC (Seq. ID No. 6), respectively. The recombinant proteins, thus, can be inducibly over-expressed by the supplement of isopropyl 13-D-1-thiogalactopyranoside (IPTG, 1 g/mL) in any Gram-negative bacteria, such as Escherichia coli BL21 (DE3). E. coli clones were cultured in Luria-Bertani (LB) broth at 37° C. for 4 hours with IPTG induction, where the original culture was 1/100 dilution with LB broth prior to IPTG induction. Because the recombinant NanA, NanB and NanC are histidine-tagged fusion proteins with the sizes of 100, 80, and 85 kDa, respectively, they may be easily purified according to the manufacturer's instructions for any kinds of Ni²⁺ affinity chromatography, such as Nickel-Chelating Resin (Invitrogen, Carlsbad, Calif., USA).

TA Exposure Activities on Cells Used to Confirm the Features of Recombinant NanA, NanB and NanC

Referred to FIGS. 3A-3D, the TA exposure activities of the recombinant neuraminidases were tested.

Lectins are usually used to recognize glycoconjugate residues (such as TA antigen) on cells. Fluorescein-labeled peanut agglutinin (PNA; Vector Laboratories, Inc., Burlingame, Calif. 94010, U.S.A.) is commonly used to detect TA on cells. Fluorescein-labeled Sambucus Nigra lectin (SNA; Vector Laboratories, Inc., Burlingame, Calif. 94010, U.S.A.) and biotinylated Maackia Amurensis lectin II (MAL II; Vector Laboratories, Inc., Burlingame, Calif. 94010, U.S.A.) are applied to recognize α2-6 and α2-3 sialyl linkages, respectively.

For the detection of the glycoconjugates on red blood cell (RBC), freshly collected blood samples from healthy volunteers were used to prepare the RBC fraction according to the method described in AABB Technical Manual, 14th Edition (http://freetechebooks.com/ebook-2011/aabb-technical-manual.html). RBC (3×10⁷ cells/mL), A549 (human epithelial lung cell line; ATCC® Number: CCL-185™) and HK-2 (human kidney 2 cell line; ATCC® Number: CRU-2190™) cells (1×10⁶ cells/mL) were cultured in Dulbecco's modified Eagle's medium (DMEM) plus Ham F12 medium, and treated with neuraminidase NanA, NanB or NanC (1 μg for RBC; 0.1 μg for A549 and HK-2). The mixture was incubated at 37° C. for 1-2 hours. For flow cytometric (FACScan, Becton Dickinson, USA) analysis, 10,000-20,000 cells were used, and cell labeling with each of lectins, including PNA, SNA and MAL II was done at 4° C. for one hour, rather than higher temperature (such as 37° C.) and longer time period (such as overnight) to cause cell agglutination, which would jam flow analysis. Biotinylated MAL II labeling can be observed by using fluorescein-conjugated streptavidin. Furthermore, to observe for cell agglutination by microscopy, 20 μl aliquots of lectin-labeled RBC were incubated at 37° C. for 30 minutes.

As shown in a previous report, TA exposure on RBC, platelets and glomeruli is mediated by the secreted NanA in pneumococcal infection. To proof whether NanC was also a potential virulence factor associated with HUS, the ability of NanC was analyzed to expose TA on cells. When RBC, A549 and HK-2 cells were treated with the recombinant NanB and NanC, TA exposure was detected (FIGS. 3A, 3B, and 3C). On RBC, the TA exposure activity of NanA had shown to reach a plateau when NanA used was more than 0.01 μg. Thus, 0.01-μg NanA was used to compare with 1-μg NanB and 1-μg NanC. The results showed that the activity of NanA was 9.4×10² and 5.3×10² times higher than those of NanB and NanC, respectively. When lectin-PNA was used to verify TA exposure on RBC, NanA-treated RBC showed larger aggregates under microscopic examination, compared to the treatments by NanB and NanC (FIG. 3D), whereas no agglutination was present with PNA in the case of untreated RBC. NanA activity shown on A549 cells was 2.2 and 3.3 times higher than NanB and NanC, respectively, while NanA activity on HK-2 cells was 1.5 times higher than both NanB and NanC.

C. Protection by Immunization Using Recombinant NanA, NanB, and NanC as Antigens

In order to develop an ideal vaccine against pneumococcal infection, particularly to be a universal protein vaccine, we chose three pneumococcal neuraminidases as a vaccine material to immunize mice, while PPV23 vaccine was used as a positive control. Meanwhile, the neuraminidase-immunized antisera were also applied for the inhibition assay against the neuraminidase activity.

Vaccination Against S. pneumoniae in Mice

Referred to FIGS. 4 and 5, the recombinant NanA, NanB and NanC were used as the antigens to protect the mice against S. pneumoniae in mice in this invention. For comparison of vaccination, the individual or combination of neuraminidases NanA, NanB and NanC (10 μg/each enzyme) were applied to immune mice (BALB/C, one month old) four times at 2-week interval prior to S. pneumoniae (3×10³ cfu) challenge, while 23-valent Pneumovax® (Merck Sharp & Dohme Corp., NJ08889, USA) polysaccharide vaccine (PPV23) and only phosphate buffered saline (PBS) were used as positive and negative controls, respectively. Freund's complete adjuvant for first time immunization and Freund's incomplete adjuvant for the last three immunizations were used with the ratio of 1:1 to the antigen(s).

To confirm the efficacy of mouse polyclonal anti-neuraminidase(s) antisera which were immunized by neuraminidase(s), we performed enzyme-linked immunosorbent assay (ELISA) which is based on the antigen-antibody sandwich principle. For ELISA test, the neuraminidase was first coated on an ELISA plate (Corning Incorporated, Corning, N.Y., USA). The anti-neuraminidase antiserum raised from mouse was then added to test how much antiserum was able to specifically bind on ELISA plate, and the antigen-antibody interaction was quantified by goat HRP-conjugated antimouse immunoglobulin G (IgG) as a secondary antibody (Millipore, Billerica, Mass. 01821, USA) and TMB/peroxide (R&D Systems, Minneapolis, Minn., USA) as a color-developing substrate. The post-immune antisera against neuraminidase(s) from different groups of combinations were tested, while the control sera from the pre-immune and the negative control with only PBS plus Freund's complete/incomplete adjuvants were also examined (FIG. 4). The value of antigen-antibody interaction was measured and presented logarithmically, as shown in FIG. 4. The results showed that each value of neuraminidase-immunized antisera, compared to the control sera, was 3-4 logarithm folds increase, revealing that each of three neuraminidases is an ideal antigen for immunization (FIG. 4).

For development of mouse vaccine against S. pneumoniae, the individual or combination of three neuraminidases, NanA, NanB and NanC with 10 μg of each enzyme, were applied to immunize nine mice (BALB/C, one month old) four times at 2-week interval, while PPV23 vaccine and a negative control (PBS+Freund's adjuvant) were taken for comparison. Thereafter, S. pneumoniae serotype 3 (3×10³ cfu) was used to challenge those immunized mice (FIG. 5). Freund's complete adjuvant for the first time immunization and Freund's incomplete adjuvant for the last three immunizations were used to mix with the antigen(s) with the ratio of 1:1. Mouse survival rate (%) was determined during 14 days of feeding after four times neuraminidase immunization and a following challenge using S. pneumoniae serotype 3. As shown in FIG. 5, vaccination tests showed that the group with the combination of three neuraminidases (NanA+NanB+NanC) presented the same 67% survival rate as that using the PPV23 vaccine, which value was the highest when compared to the other groups with one or two of three neuraminidases. In this invention, the combination containing three neuraminidases (NanA, NanB, and NanC) together was evaluted to be the best vaccine candidate for vaccination against S. pneumoniae infection in mice, and it also would be an appropriate candidate as a kind of universal protein vaccine.

Inhibition of Neuraminidase Activity by Antibodies or Immunized Antisera

As referred to FIGS. 6A to 7C, the inhibition assay was taken to test how efficient the neuraminidase activity can be inhibited by neuraminidase-specific antisera. The antisera against individual neuraminidase (NanA, NanB or NanC) were raised from rabbits, and then tested for their inhibitory effect on the activities of neuraminidases. NanC antiserum was purified by Protein A Sepharose beads (GE Healthcare) to increase its inhibition efficiency. Different amounts of neuraminidases in 10-μL PBS were pre-incubated with 10-μL immunized serum for 5 minutes. The antiserum-treated neuraminidase was mixed with RBC cells (4×10⁶ cells/mL) for 2 hours of incubation at 4° C. Inhibition of neuraminidases (NanA, NanB, and NanC) activity by rabbit antisera was quantified by TA exposure on RBCs using flow cytometry, wherein the detection of TA exposure on RBC cells using FITC-labeled PNA lectin was described in previous section. If neuraminidase is neutralized by a specific anti-neuraminidase serum, the exposure of TA antigen on RBC cells, and the value of fluorescence intensity will be reduced.

As shown on y-axis of FIGS. 6A-6C, the activities of NanB and NanC for TA antigen exposure were naturally weaker than that of NanA. NanA activity was completely inhibited by 30-μg anti-NanA antiserum when both 0.1-μg and 0.01-μg NanA were used, while only 20% activity was inhibited when 1-μg NanA was used (FIG. 6A). The 1-μg NanB activity was completely inhibited by 30-μg anti-NanB antiserum. However, only 40% NanC activity was inhibited by 30-μg anti-NanC antiserum, but 90% NanC activity was inhibited by 30-μg purified anti-NanC antiserum (FIG. 6C).

For cross-reactivity, antineuraminidase (including NanA, NanB and NanC) antisera were tested. Anti-NanA antiserum did not show inhibitory effect on the activity of NanB and NanC and vice versa. However, anti-NanB antiserum could inhibit NanC activity by 74%. Non-purified and purified anti-NanC antisera inhibited NanB activity by 40% and 76%, respectively.

The antisera from rabbit used to inhibit 50% neuraminidase activity were also assessed by titration test (data not shown). The dilution of anti-NanA antiserum for 50% NanA inhibition was 1, 8, and 32 for 1 μg, 0.1 μg and 0.01 μg, respectively. 50% NanB (1 μg) activity with anti-NanB antiserum was inhibited by 16 folds of dilution. The dilutions of non-purified and purified anti-NanC antisera to inhibit 50% NanC (1 μg) activity were 1 and 8, respectively.

Similar to the rabbit antineuraminidase antisera, the antineuraminidase antisera raised from mouse were also shown to specifically inhibit neuraminidase activities, as shown in FIGS. 7A-7C. The 30-μg mouse Anti-NanA and anti-NanB antisera enable to completely inhibit the activities of 0.1-μg NanA and 1-μg NanB, respectively. However, anti-NanC antiserum (30-μg) was only able to neutralize 50% NanC (1-μg) activity.

Taken together, the results indicated that each of anti-neuraminidase antisera raised from both rabbit and mice enables to specifically inhibit or neutralize its corresponding neuraminidase activity; however, only anti-NanB and anti-NanC antisera have cross-protection abilities against each other. Although the inhibition of the anti-NanC antiserum was not as efficient as those anti-NanA and anti-NanB antisera, the combination of NanC with NanA and NanB is the best candidate as a vaccine.

Having described the invention by the description and illustrations above, it should be understood that these are exemplary of the invention and are not to be considered as limiting. Accordingly, the invention is not to be considered as limited by the foregoing description, but includes any equivalents. 

What is claimed is:
 1. A method of providing protection against pneumococcal infection in a subject comprises steps of: administering immunogenic amount of composition that includes the combination of three recombinant pneumococcal neuraminidases: NanA, NanB, and NanC of Streptococcus pneumoniae to the subject; wherein the recombinant pneumococcal neuraminidase NanA is SEQ ID NO: 4; wherein the recombinant pneumococcal neuraminidase NanB is SEQ ID NO: 5; wherein the recombinant pneumococcal neuraminidase NanC is SEQ ID NO: 6; wherein administering the recombinant pneumococcal neuraminidases elicits an immune response to Streptococcus pneumoniae.
 2. The method of providing protection against pneumococcal infection in a subject of claim 1, further comprises a step of adding adjuvants to enhance the immune response.
 3. The method of providing protection against pneumococcal infection in a subject of claim 1, wherein said Streptococcus pneumoniae includes all kinds of Streptococcus pneumoniae serotypes.
 4. The method of providing protection against pneumococcal infection in a subject of claim 1, wherein the subject is human.
 5. The method of providing protection against pneumococcal infection in a subject of claim 1, wherein the subject is animal. 